Contamination of soils and ground water with toxic, environmentally persistent chemicals is a serious, ongoing problem. Toxic, environmentally persistent chemicals are those that are resistant to degradation in the natural environment. As such, these chemicals pose a multifaceted problem in that as they persist and accumulate in the environment, their toxicity, including in many instances, proven carcinogenicity, presents substantial health risks to both animals and human beings. The medical literature is full of data on the adverse health effects caused by human exposure to these chemicals.
The scope of this problem can be appreciated when one considers that there are thousands of toxic waste sites in this country where large quantities of these chemicals either have been dumped directly into the soil or have leaked out of storage tanks. The disposal of contaminated materials, e.g., storage containers, manufacturing equipment, and chemically treated materials such as lumber, presents an additional problem.
The types of chemicals that fit the classification of a toxic, environmentally persistent pollutant, and therefore contribute to this problem, are numerous. One such type is the halogenated aromatic compounds (HAC's). HAC's are further categorized into those whose molecular structure has a single aromatic ring and those whose structure contain two or more. The HAC's that contain a single ring include pentachlorophenol (PCP). PCP has been used extensively as a wood preservative. The HAC's containing two or more rings include the pesticide, DDT, and polychlorinated biphenyls and polybrominated biphenyls that have been used extensively in manufacturing.
The prior art is replete with laboratory data on methods for the degradation of hazardous chemicals, generally, and specifically, of HAC's. The difficulty with many of these methods is that while they may be successful in controlled laboratory or controlled field conditions, they often lack effectiveness or practicality in large scale field conditions, i.e., they are less than successful when tried with "field-contaminated" materials. As used herein, the term "field-contaminated" is meant to refer to a solid material, i.e., soil or wood, that has been contaminated through use or accident as compared to laboratory samples that are artificially contaminated, particularly under controlled laboratory conditions. In the case of wood products, "field-contaminated" also is meant to refer to a wood that has been contaminated in the field, a wood that has been intentionally treated for use in commerce, or a wood that is a contaminated by-product of industrial treatment. An example of a wood which has been intentionally treated for use in commerce is wood products which have been treated with a material that contains hazardous chemicals, such as a wood preservative.
Treatment strategies that have been suggested or tried include incineration of the waste, removal and isolation of the contaminated materials, and degradation of the pollutant by bacteria. All of these strategies suffer from serious deficiencies. Incineration is extremely expensive due to the energy requirements and the necessity of hauling the contaminated material to remote locations, as well as impractical due to the large quantities of waste that need to be processed. Removal and isolation of the contaminated material is also expensive and does nothing to effect a long term solution. Degradation of the chemicals by bacteria has proven less than ideal due to the bacteria's specificity for particular chemicals and sensitivity to the toxic chemicals and environmental conditions.
Another potential strategy that has been the subject of experimental research in the past decade is the use of a class of wood degrading fungi, known as lignin-degrading fungi, to degrade HAC's. In the early 1980's, it was reported that the fungus Phanerochaete chrysosporium (P. chrysosporium) could degrade chlorinated organics in the effluent of a kraft pulp mill (Eaton et al., TAPPI, vol. 65 (1982) pp. 89-92; Huynh et al., TAPPI, vol. 68 (1985) pp. 98-102). At about this same time, other researchers isolated and characterized the enzymes responsible for the lignin degrading ability of these fungi. These isolated enzymes, termed lignin peroxidases or ligninases, were found to oxidize a wide variety of compounds in addition to lignin. Such compounds include polycyclic aromatic hydrocarbons (Hammel et al., J. Biol. Chem., vol. 261 (1986) pp. 16948-16952; Sanglard et al., Enzyme and Microbial Tech., vol. 8 (1986) pp. 209-212), dibenzo(p)dioxins (Hammel et al., J. Biol. Chem. , vol. 261 (1986) pp. 16948-16952); Haemmerli et al., J. Biol. Chem., vol. 261 (1986) pp. 6900-6903), and polychlorinated phenols (Hammel and Tardone, Biochem., vol. 27 (1988) pp. 6563-6568).
It has since been reported that under controlled laboratory conditions, cultures of P. chrysosporium can also effectively degrade HAC's. In particular, Bumpus et al. have extensively studied the degradation of the multi-ring halogenated aromatics. They have shown that small cultures of P. chrysosporium grown in the laboratory in a low nitrogen-containing growth medium at approximately 37.degree. C. will degrade DDT, 2,4,5,2',4',5'-hexachloro-biphenyl, 2,3,7,8-tetra-chlorodibenzo-p-dioxin, and lindane (see, Bumpus et al., Science, vol. 228 (1985) pp. 1434-1436; Bumpus and Aust, Applied and Environmental Microbiology, vol. 53 (1987) pp. 2001-2007; Aust and Bumpus, U.S. Pat. No. 4,891,320). Similar studies have analyzed the ability of P. chrysosporium to degrade PCP in controlled small cultures (Mileski et al., Applied and Environmental Microbiology, vol. 54 (1988) pp. 2885-2889).
Far fewer studies have attempted to more closely approximate conditions that are present at contaminated field sites or investigate the relative efficacies of the many other species of fungi of the lignin-degrading class. Several studies have reported that P. chrysosporium can degrade 2,4,5-trichlorophenoxyacetic acid (Ryan et al., Appl. Microbiol, Biotechnol., vol. 31 (1989) pp. 302-307), fluorene (George and Neufeld, Biotechnol. Bioeng., vol. 33 (1989) pp. 1306-1310), and PCP (Lamar et al., Soil Biol. Biochem., vol. 22 (1990) pp. 433-440) in chemically spiked sterile soil in laboratory culture. This latter study also reported that the ability of P. chrysosporium to degrade the PCP varied depending on the soil type; the fungi exhibited the highest rate of transformation of the PCP in Marshan soil (sandy loam) and the lowest in Batavia soil (silty clay loam).
Lamar et al., Appl. Environ. Microbiol., vol. 56 (1990) pp.3519-3526, also tested seven species of lignin-degrading fungi in laboratory cultures. They reported significant differences between the species in both the rate and extent of degradation of the PCP. Most recently, in a preliminary field study in 1 m.sup.2 plots of soil contaminated exclusively with PCP, the PCP degrading abilities of P. chrysosporium and Phanerochaete sordida (P. sordida) were compared (Lamar et al., Appl. Environ. Microbiol., vol. 56 (1990) pp. 3093-3100). The results showed that in the very alkaline soil (pH approximately 9.6) that had been tilled prior to initiation of the study to allow evaporation of mineral spirits, sterilized, and supplemented with peat moss, both fungi efficiently degraded the PCP over a 45 day period.
Although these laboratory and preliminary field study data are useful in defining a promising technology for the degradation of HAC's, such a technology is useless if it will not work effectively at the large scale needed to clean up the vast quantities of soils and materials contaminated with HAC's. As noted previously, many technologies that were thought to hold great promise for solving a problem did not prove effective when put to a large scale test. One such example was the technology of degradation of the chemical PCB's with certain genetically engineered or adapted bacteria. Results showed that under controlled laboratory conditions, successful degradation was effected. However, when field trials were conducted, the bacteria were found to be too sensitive to the varying environmental conditions for effective use. (R. Unterman, "Bacterial Treatment of PCB-Contaminated Soils," Hazardous Waste Treatment by Genetically Engineered or Adapted Organisms, p. 17, Nov. 30-Dec. 2, 1988, Washington, D.C.)
The problems that are encountered when scaling up a technology are numerous. In large scale use, the parameters controlled in the laboratory are uncontrolled. For example, in the laboratory, parameters such as the temperature, humidity, concentration and distribution of the pollutant chemical, purity of the chemical, oxygen (O.sub.2) content, pH, and organics/nutrient content of the cultures can be controlled. However, in large scale use, the temperature and humidity will constantly fluctuate. The concentration and distribution of the pollutant chemical will vary in different areas of the contaminated site. The site will be contaminated with other chemicals, The O.sub.2 content will depend on such things as water content, the particle size of the soil or contaminated material, and how tightly it is packed. The pH and organics/nutrient content will depend in part on the geology and nature of the dump site.
Compounding these problems are unknowns in terms of what effect the total environment will have on fungi physiology. The environment as a whole (including the parameters listed above as well as others) affects not only the growth rate of the fungi, but also the stimulation/repression of their enzymatic systems. For example, in the laboratory, the lignin peroxidases of P. chrysosporium are induced under conditions of low nitrogen and repressed under conditions of high nitrogen (Fenn and Kirk, Arch. Microbiol., vol. 130 (1981) pp. 59-65), but the lignolytic enzymes of other studied species of fungi are not regulated in this manner. Also, the ability of lignin-degrading fungi to degrade HAC's may not depend entirely on enzymes of the lignolytic system (Kohler et al., Appl. Microbiol. Biotech., vol. 29 (1988) pp. 618-620), and under identical laboratory conditions, the lignolytic enzymes expressed by P. chrysosporium and P. sordida differ (R. Lamar, unpublished data). As this sparsity of data indicates, virtually nothing is known about how particular environmental and contamination site conditions influence the enzymatic pathways and/or growth of individual fungi, or how effectively or ineffectively the enzyme systems of different fungal species will function in degrading HAC's in uncontrolled field conditions. Consequently, the outcome of full scale field trials cannot be predicted.
Thus, the prior art teaches that lignin-degrading fungi can degrade HAC's in controlled laboratory experiments, and degrade PCP in soil in a controlled small scale field test. However, the prior art does not teach the methods or specific fungi required to successfully employ lignin-degrading fungi technology for use in the full scale bioremediation of halogenated hydrocarbon-contaminated, particularly PCP-contaminated soils and other materials.